Zinc finger transcription factor ZFP1 is associated with growth, conidiation, osmoregulation, and virulence in the Polygonatum kingianum pathogen Fusarium oxysporum

Rhizome rot is a destructive soil-borne disease of Polygonatum kingianum and adversely affects the yield and sustenance of the plant. Understanding how the causal fungus Fusarium oxysporum infects P. kingianum may suggest effective control measures against rhizome rot. In germinating conidia of infectious F. oxysporum, expression of the zinc finger transcription factor gene Zfp1, consisting of two C2H2 motifs, was up-regulated. To characterize the critical role of ZFP1, we generated independent deletion mutants (zfp1) and complemented one mutant with a transgenic copy of ZFP1 (zfp1 tZFP1). Mycelial growth and conidial production of zfp1 were slower than those of wild type (ZFP1) and zfp1 tZFP1. Additionally, a reduced inhibition of growth suggested zfp1 was less sensitive to conditions promoting cell wall and osmotic stresses than ZFP1 and zfp1 tZFP1. Furthermore pathogenicity tests suggested a critical role for growth of zfp1 in infected leaves and rhizomes of P. kingianum. Thus ZFP1 is important for mycelial growth, conidiation, osmoregulation, and pathogenicity in P. kingianum.

The F. oxysporum homolog of the TF Ste12 which possesses a C 2 H 2 domain, was up-regulated during the infection process, and was necessary for F. oxysporum virulence 16 .On the contrary, the pH signalling TF PacC with a C 2 H 2 domain negatively regulated virulence, preventing the transcription of acid-expressed genes essential during F. oxysporum infection 17 .Zinc homeostasis regulator ZafA is also a C 2 H 2 ZF.It was significantly upregulated during the early stages of infection and was required for the full virulence of F. oxysporum, especially when zinc was limited 18 .However, FolCzf1, a C 2 H 2 ZF in F. oxysporum f. sp.lycopersici (Fol), was required for growth, conidiation, conidia morphology, and pathogenicity in tomato 19 .Similarly, the Con7-1 (a C 2 H 2 ZF in F. oxysporum) deletion mutant exhibited defects in chitin synthase, hyphal branch, conidiation, and virulence 20 .Although these five C 2 H 2 ZFs have been characterized in F. oxysporum, the function and regulation of most ZFs remain to be studied.
In this study, we identified ZF protein TF ZFP1 with two C 2 H 2 domains, a homologue of Fol 4287 ZF protein MSN2/4 21 , whose function was still unknown.Therefore, this study aimed to investigate the roles of ZFP1 in the developmental processes and pathogenicity of the F. oxysporum in P. kingianum.The mutant zfp1 and zfp1 tZFP1 were generated by the target gene replacement technique.The gene Zfp1 was up-regulated during F. oxysporum conidial germination.The inhibition rates, sensitivity under cell wall and osmotic targeted stresses, and virulence of zfp1 were decreased compared to those of wild type ZFP1 and zfp1 tZFP1.The results elaborated the effects of ZFP1 on the growth, conidiation, stress response, and virulence of F. oxysporum, which detected an important gene that can be silenced using host-induced gene silencing for the prevention and control of the disease in the near future.

Assessment of Zfp1 expression pattern during conidial germination
The transcriptional induction of Zfp1 in germinating conidia was previously observed in published analysis of RNA sequencing 22 .To further assess the expression pattern of Zfp1 during conidial germination, RNA was extracted at 0 h, 12 h, and 24 h and analyzed using RT-qPCR.The expressions of Zfp1 in transcriptome data and RT-qPCR results were highly identical.The result indicated that expression of Zfp1 was up-regulated in germinating conidia of infectious F. oxysporum (Fig. 1).

ZFP1 is a C 2 H 2 -type ZF protein
To analyze the structure of ZFP1, we performed BLASTn analysis in NCBI, and compared the deduced amino acid sequences of several ZF proteins by phylogenetic tree construction and multiple alignment.The gene length of Zfp1 (GenBank accession no.OR715798), identified from genome sequence resource of F. oxysporum PkF01 (GenBank assembly accession no.JAMBQE000000000) 23 , was 1,836 bp; it contains one intron, and the length of coding sequence (CDS) was predicted to be 1,596 bp, encoding 531 amino acid residues.BLASTn analysis showed that Zfp1 nucleotide sequence was 99% identity to the ZF gene MSN2/4 of Fol 4287 (FOXG_01955, GenBank accession no.XM_018379112).Additionally, a phylogenetic tree constructed based on the predicted amino acid sequences showed that ZFP1 (protein_id WOW16306) was classified into the C 2 H 2 -type subfamily, Ste12 (protein_id ACM80357) 16 (Fig. 2A), and multiple alignment of predicted amino acid sequences showed that ZFP1 contained two C 2 H 2 zinc finger domains (Fig. 2B), which making it a C 2 H 2 -type ZF protein.

Generation of deletion mutant zfp1 and its complementation by a transgenic copy of ZFP1
To study the critical roles of ZFP1, we generated three independent deletion mutants (zfp1-1, zfp1-2 and zpf1-3) by targeting Zfp1 for gene replacement (as depicted in Fig. S1).Two DNA fragments were introduced into F. oxysporum using genetic transformation, one was sequence upstream of ZFP1 coding sequence fused with a partial hygromycin resistance gene, while the other was an overlapping partial hygromycin resistance gene fused to sequence downstream of ZFP1 coding sequence.Homologous recombination of the two DNA fragments and the ZFP1 genomic locus was expected to create a full-length hygromycin resistance gene that replaced coding sequence of ZFP1.
The correct gene replacement was verified using PCR, which showed that coding sequence of ZFP1 was successfully replaced in the hygromycin B-resistant transformants.Coding sequence of ZFP1 (942 bp) could be PCR-amplified from wild type ZFP1 and not from zfp1 mutants (Fig. 3A).Instead in the mutants three fragments of expected sizes (1,597 bp, 1,100 bp, 1,216 bp), corresponding to the full-length hygromycin resistance gene and fusions of hygromycin resistance gene to sequences upstream and downstream of ZFP1, were amplified respectively (Fig. 3A).One mutant zfp1 was complemented by integration of an ectopic copy of wild type ZFP1.Coding sequence of ZFP1 (942 bp) could be PCR-amplified from DNA of ZFP1 and zfp1 tZFP1 and not zfp1 mutants (Fig. 3B).A hygromycin-resistance gene was PCR-amplified from the zfp1 and zfp1 tZFP1 transformant and neither from ZFP1 (Fig. 3C).A neomycin-resistance gene, which was included in the transgenic construct and used to select for stable integration, was only PCR-amplified from the zfp1 tZFP1 transformant and neither from ZFP1 nor zfp1 (Fig. 3D).Expression of Zfp1 was significantly reduced in zfp1 mutants and restored in zfp1 tZFP1 (Fig. 3E).These results validated the loss of ZFP1 in zfp1 mutants and transgenic complementation of zfp1 in zfp1 tZFP1.

Deletion of Zfp1 affects mycelial growth and conidial formation
To investigate the functions of Zfp1 deletion in vegetative growth and asexual reproduction, we determined growth rates and conidiation of wild type ZFP1, zfp1 and zfp1 tZFP1.The zfp1 colony formation and growth rates were significantly reduced compared to ZFP1 and zfp1 tZFP1 cultured on potato dextrose agar (PDA) for 5 days (Fig. 4A, B).Furthermore, a significantly reduced number of micro-conidia was produced in zfp1 from 48 to 96 h post-inoculation on potato dextrose broth (PDB) (Fig. 4C).However, zfp1 conidial morphology was not different compared to ZFP1 and zfp1 tZFP1 at 4 h incubation in PDB (Fig. 4D).These results indicated that deletion of Zfp1 affected mycelial growth and conidial formation in F. oxysporum.

Deletion of Zfp1 reduces virulence
To investigate the effects of Zfp1 deletion on virulence, we inoculated detached leaves, detached rhizomes, and whole plants with conidial suspension of wild type ZFP1, zfp1 and zfp1 tZFP1.In the infection assays, the zfp1 lesion diameters in the detached leaves and rhizomes were significantly decreased compared to ZFP1 and zfp1 tZFP1 (Fig. 5A, B).Withering of the leaves and rhizome rot were observed among the P. kingianum plants inoculated with ZFP1 and zfp1 tZFP1.However, no symptoms were observed in zfp1 (Fig. 5C-E).Overall, the disease index of ZFP1 and zfp1 tZFP1 was significantly higher than that of zfp1 (Fig. 5F).These results demonstrated that zfp1 strongly reduced the virulence of F. oxysporum on P. kingianum.

ZFP1 contributes to F. oxysporum stress responses
To investigate the effects of ZFP1 on F. oxysporum adaptation to infection-related stresses, we compared the radial growth rates of wild type ZFP1, mutant zfp1, and zfp1 tZFP1 on PDA under cell wall stresses (congo red, CR; sodium dodecyl sulfate, SDS), oxidative stress (H 2 O 2 ), osmotic stresses (NaCl, KCl), tebuconazole and carbendazim stresses.The inhibition rates and sensitivity of zfp1 under CR, SDS, NaCl, KCl, and tebuconazole stresses were decreased compared to ZFP1 and zfp1 tZFP1.However, zfp1 had a lower tolerance to carbendazim compared to ZFP1 and zfp1 tZFP1.There was no significant difference in the inhibition rates of ZFP1, zfp1, and zfp1 tZFP1 under H 2 O 2 , suggesting that ZFP1 had little effect on the sensitivity to oxidative stress (Fig. 6).These results suggested that ZFP1 was involved in regulating responses to cell wall integrity stresses and osmotic pressure in F. oxysporum.

Discussion
Rhizome rot is a devastating soil-borne disease, seriously threatening the P. kingianum industry.F. oxysporum invades the roots, causing wounds, colonizes the vascular tissues, blocking water and nutrient transport, and may even lead to plant death 24 .Although over 700 TFs have been predicted in F. oxysporum, only 15 ZFs are associated with pathogenicity 15 .Among them is Fow2, a Zn(II) 2 Cys 6 -type transcription regulator, essential for root invasion and colonization but not for vegetative growth and conidiation in F. oxysporum 25 .The cutinase transcription factors ctf1 and ctf2, containing the Zn 2 Cys 6 DNA binding domain, also play important roles in the lipolytic system of Fol.Additionally, ctf1 and ctf2 deletion mutants severely reduce F. oxysporum virulence 26 .
In common bean, Fusarium transcription factor 1 (ftf1) with a Zn(II) 2 Cys 6 motif is only up-regulated during plant infection, where multiple ftf1 copies increase virulence in F. oxysporum f. sp.phaseol 27 .Additionally, EBR1, belonging to the Zn 2 Cys 6 family, regulates the expression of genes encoding metabolism and virulence.EBR1 deletion impairs growth and reduces pathogenicity and biocontrol capacities in different F. oxysporum strains 28 .Furthermore, the global nitrogen regulator FNR1, with a single conserved GATA-type ZF domain, regulates the secondary nitrogen acquisition in plants.Notably, the disruption of FNR1 mutants significantly delays F. oxysporum infection in tomato seedlings 29 .Besides, Cti6, which contains a PHD finger motif and simultaneously interacts with the transcriptional corepressor complex Cyc8-Tup1 and the co-activator SAGA (Spt-Ada-Gcn5acetytransferase) complex, is required for full virulence in F. oxysporum on tomato 30 .In this study, we identified C 2 H 2 ZF ZFP1 (protein_id WOW16306) was 88%, 57%, and 46% identity with F. graminearum GzC2H045 (pro-tein_id XP_011326528) 31 , Verticillium dahliae VdMsn2 (protein_id XP_009648969) 32 , and Magnaporthe oryzae MoMSN2 (protein_id MGG 00,501) 33 , respectively.Disruption of these three genes all had defects on hyphal growth and virulence, which were similar to those of zfp1.However, the regulatory mechanism in pathogenicity needs to be further analyzed in the plant-pathogen interaction, which includes investigation the proteins interacting with ZFP1 by yeast two-hybrid technique, prediction the location of Zfp1 by subcellular location, and detection the differences of fusarium acid content between wild type ZFP1 and mutant zfp1, etc.The host immune system first detects the pathogenic fungi conidia during the infection process.With F. oxysporum, conidial germination is the key step of infection 34 .Therefore, early conidial detection is crucial to inhibit fungal growth and alleviate the disease occurrence 35 .In this study, C 2 H 2 ZF Zfp1 was screened using the transcriptome data of the conidial germination process in F. oxysporum 22 .Zfp1 was significantly up-regulated during conidial germination, suggesting that this gene might be related to F. oxysporum growth and pathogenicity.To verify this conjecture, zfp1 and zfp1 tZFP1 were constructed using split-marker homologous recombination, which revealed that ZFP1 regulated mycelial growth and conidial yield and thus the virulence of F. oxysporum.However, this did not affect the conidia morphology.Similarly, BcTaf14, TATA box-binding protein-associated factor 14 (Taf14) in Botrytis cinerea was associated with mycelial growth, conidiation, and conidial morphogenesis with no effect on conidial germination 36 .snt2, a PHD-containing ZF, was also essential in vegetative growth, conidial production, and host colonization by F. oxysporum f. sp.melonis 37 .However, the white-collar 1 photoreceptor Wc1, a GATA ZF, played roles in the hyphae development of F. oxysporum, but was dispensable for pathogenicity on tomato plants 38 .When a pathogen invades the host, the host exhibits some defensive response, including a change in the internal environment or vascular system 39 .Similarly, pathogenic fungi always adopt a series of complex strategies for successful invasion of the host, including the plant defense mechanisms, host intracellular environment, and defeating adverse environmental changes 40,41 .In this study, zfp1 increased resistance to membrane stressor (SDS), cell wall stressor (CR), extracellular osmotic (NaCl), and intracellular osmotic (KCl) stress compared to the ZFP1.However, no differences were identified in tolerance to oxidative (H 2 O 2 ) stress between the mutants zfp1 and the wild type ZFP1, implying that ZFP1 regulated osmotic pressure and cell wall integrity stresses.These results are similar to those of Aoime2 (Arthrobotrys oligospora inducer of meiosis 2) deletion mutants which were unaffected by oxidative stressor H 2 O 2 but highly sensitive to the osmotic stressor NaCl 42 .Similarly, BcTaf14 deletion mutants increased NaCl and KCl sensitivity 36 .Neutral trehalase-encoding gene NTH1 knockout mutant was also sensitive to H 2 O 2 and SDS but not to CR, NaCl, and KCl 43 .On NaCl plates, there was almost no radial growth of zfp1 and ZFP1.Interestingly, growth of zfp1 and ZFP1 appeared to be more comparable on plates with KCl, suggesting that KCl was suppressing the defect of zfp1.www.nature.com/scientificreports/ In conclusion, the C 2 H 2 ZF protein ZFP1 plays significant roles in mycelial growth, conidiation, stress response, and virulence in F. oxysporum of P. kingianum.However, how ZFP1 exerts these functions requires further study.

Isolation of the fungal strain and the culture conditions
The wild-type F. oxysporum strain PkF01 isolated from the P. kingianum rhizome rot samples, was identified with nucleotide sequences of the elongation factor 1-alpha (GenBank accession no.MW149127) and the second largest subunit of nuclear DNA-directed RNA polymerase II (GenBank accession no.MW194100) by L. Zhang in our previous study 3 .For conidia production, mycelia were incubated in PDB at 28 °C with shaking at 180 revolutions per minute (rpm) for 3 days.Subsequently, the conidial suspension was adjusted to 1 × 10 6 conidia•mL -1 , and 30% glycerol was added before storing the suspension at − 80 °C44 .

Phylogenetic tree construction and protein sequence alignment
Zfp1 nucleotide sequence was submitted to the National Center for Biotechnology Information (NCBI) Gen-Bank database, and BLASTn analysis was performed in NCBI.To further investigate the function of ZFP1 in F. oxysporum, phylogenetic tree and multiple alignment of the C 2 H 2 zinc-finger proteins of F. oxysporum were performed using the maximum likelihood method with 1,000 replications of bootstrap in MEGA 11 45 and edited in GeneDoc 46 , respectively.

Complementation of Zfp1 deletion mutant (zfp1 tZFP1)
Zfp1 was complemented with a 1,848 bp fragment containing the complete open reading frame of Zfp1.The fragment amplified with Zfp1-CF/Zfp1-CR primer pair was cloned in frame with the strong constitutive Aspergillus nidulans gpdA promoter contained in vector pDHtsk-GFP-G418 with a neomycin-resistant cassette.Next, the recombinant plasmid was transformed into zfp1 protoplasts.Subsequently, the transformants were screened on PDA containing hygromycin B (400 mg•L −1 ) and neomycin (300 mg•L −1 ).

Verification and quantification of gene expression
The deletion mutants were verified by PCR using primer pair Zfp1-IF/Zfp1-IF, Zfp1-UH-F/Zfp1-UH-R, and Zfp1-DY-F/Zfp1-DY-R.The complementary mutants were verified using Zfp1-IF/Zfp1-IR, Hy-F/Yg-R, and Neo-F/Neo-R primer pairs.RT-qPCR further validated Zfp1 expression.RNA extraction, cDNA synthesis, and RT-qPCR were performed with TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa, Code No. 9767), PrimeScript™ RT Master Mix (TaKaRa, Code No. RR036A), and TB Green® Premix Ex Taq™ II (TaKaRa, Code No. RR820A) according to manufacturer's instructions, respectively.The RT-qPCR conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and annealing at 60 °C for 30 s. Elongation factor 1-alpha (EF1α) and tubulin 2 (TUB2) were used as internal reference genes 51 .The relative expression of the target gene was calculated by the 2 -△△Ct method 52 .The Zfp1 gene expression were quantified by RT-qPCR using primer pair Zfp1-QF1/Zfp1-QR1, EF1α-QF/EF1α-QR, and TUB2-QF/TUB2-QR.The RT-qPCR assay was conducted with three independent biological and three technical replicates.All primers used in this study were listed in Table S1 and Fig. S1.

Mycelial growth and conidiation assays
For mycelial growth, a 5-mm-diameter mycelial plug from a 3-day-old culture was placed on PDA and incubated at 28 °C for 5 days.For 5 days, the colony morphology was photographed, and the colony diameter was measured daily.For conidiation, a 5-mm-diameter mycelial plug from a 3-day-old culture was placed in PDB (200 mL) with shaking at 180 rpm and 28 °C.The conidial yield was calculated at 48, 72, and 96 h post-inoculation in PDB using a haemocytometer.Subsequently, conidia were filtered through two layers of lens paper and resuspended to a concentration of 1 × 10 6 conidia•mL -1 in PDB.The conidial morphology was observed after 4 h of growth.The assays were performed with three biological replicates.

Pathogenicity assays
Polygonatum kingianum plants collected from plantation in Kunming city of Yunnan province, China, and were permitted and identified by P. Ji from Institute of Medicinal Plant Cultivation.One drop of conidial suspension (1 × 10 6 conidia•mL -1 ) was dripped onto the surface of each P. kingianum leaf and tuber 53,54 .Leaves/rhizomes inoculated with sterile water were used as the controls.Inoculated leaves and rhizomes were cultured on moist

Figure 1 .
Figure 1.Zfp1 expression pattern at 0, 12, and 24 h of conidia germination.Error bars represent standard deviation of the mean with three independent biological replicates.TPM means transcripts per kilobase of exon model per million mapped reads.

Figure 2 .
Figure 2. Phylogenetic relationships among F. oxysporum ZFs.(A) Phylogenetic tree constructed based on predicted amino acid sequences.Heat shock factor (HSF)-type TF was used as the outgroup.(B) Multiple alignment of predicted amino acid sequences of the C 2 H 2 ZF region of F. oxysporum.Same amino acids were marked in black, and similar amino acids were shaded in grey.Black lines indicate the two C 2 H 2 zinc-finger domains.